title: Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation.
abstract: Protease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with β-strand 3A and the loop connecting α-helix A with β-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1's protease inhibitory activity.
authors.forename: Tina M
authors.initials: TM
authors.lastname: Kousted
authors.forename: Karsten
authors.initials: K
authors.lastname: Skjoedt
authors.forename: Steen V
authors.initials: SV
authors.lastname: Petersen
authors.forename: Claus
authors.initials: C
authors.lastname: Koch
authors.forename: Lars
authors.initials: L
authors.lastname: Vitved
authors.forename: Maja
authors.initials: M
authors.lastname: Sochalska
authors.forename: Céline
authors.initials: C
authors.lastname: Lacroix
authors.forename: Lisbeth M
authors.initials: LM
authors.lastname: Andersen
authors.forename: Troels
authors.initials: T
authors.lastname: Wind
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
authors.affiliation: Jan K. Jensen, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark, E-mail: jkj@mb.au.dk.
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000915:Antibody Affinity; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry; D011487:Protein Conformation; D021381:Protein Transport; D058258:Serpin E2 / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; C545486:SERPINE2 protein, human; D058258:Serpin E2; D006493:Heparin; D010447:Peptide Hydrolases
keywords: Cancer; SERPIN E2; glia-derived nexin; inhibitory antibody; serine protease